The goal of this work is identification of a biochemical function of calretinin (CR). The results following basic characteristics of CR have been revealed: (1) CR undergoes a calcium-dependent conformational change. This was shown in several ways. First, changes in the intrinsic fluorescence (due to Trp) of CR and in the fluorescence of a probe attached to Cys residues on the protein were observed as a result of Ca2+ binding to CR. Secondly, different tryptic cleavage fragments of CR were obtained in the presence and absence of calcium as shown by identification of fragments using a combination of mass spectometry and amino acid analyses. The mass spectroscopic analyses also revealed that native rat CR is blocked at the N-terminus by a relatively small functional group. Fragments of recombinant CR synthesized in E. coli revealed similar locations of calcium-sensitive and insensitive domains on the protein. (2) Subcellular fractionation of rat cerebellum and examination of samples of radioimmunoassay or immunoblots revealed separate patterns of localization for the three calcium binding proteins, CR, calbindin D28K and parvalbumin. Both calbindin D28k and CR were associated, to a small degree, with synaptic and microsomal membranes with partial calcium dependence of this binding while parvalbumin was only detected in the cytosolic fraction. Calbindin D28k was particularly enriched in the microsomal fraction. (3) Examination of formalin-fixed and unfixed samples revealed a preferential recognition of calcium binding protein antibodies for proteins in the calcium-bound forms. Polyclonal antibodies specific to CR and monoclonal antibodies to parvalbumin and calbindin D28k recognized Ca2+-bound forms (fixed or unfixed) to a greater extent than the Ca2+-free forms. A polyclonal antibody made to a different portion of the CR molecule showing less calcium-dependent conformational changes did not show a preference for any calcium binding state of CR.